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  A mass spectrometry workflow for measuring protein turnover rates in vivo.

Alevra, M., Mandad, S., Ischebeck, T., Urlaub, H., Rizzoli, S. O., & Fornasiero, E. F. (2019). A mass spectrometry workflow for measuring protein turnover rates in vivo. Nature Protocols, 14(12), 3333-3365. doi:10.1038/s41596-019-0222-y.

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Item Permalink: http://hdl.handle.net/21.11116/0000-0005-3A8E-B Version Permalink: http://hdl.handle.net/21.11116/0000-0006-0F4D-5
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Alevra, M., Author
Mandad, S.1, Author              
Ischebeck, T., Author
Urlaub, H.1, Author              
Rizzoli, S. O., Author
Fornasiero, E. F., Author
Affiliations:
1Research Group of Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Max Planck Society, ou_578613              

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 Abstract: Proteins are continually produced and degraded, to avoid the accumulation of old or damaged molecules and to maintain the efficiency of physiological processes. Despite its importance, protein turnover has been difficult to measure in vivo. Previous approaches to evaluating turnover in vivo have required custom labeling approaches, involved complex mass spectrometry (MS) analyses, or used comparative strategies that do not allow direct quantitative measurements. Here, we describe a robust protocol for quantitative proteome turnover analysis in mice that is based on a commercially available diet for stable isotope labeling of amino acids in mammals (SILAM). We start by discussing fundamental concepts of protein turnover, including different methodological approaches. We then cover in detail the practical aspects of metabolic labeling and explain both the experimental and computational steps that must be taken to obtain accurate in vivo results. Finally, we present a simple experimental workflow that enables measurement of precise turnover rates in a time frame of ~4-5 weeks, including the labeling time. We also provide all the scripts needed for the interpretation of the MS results and for comparing turnover across different conditions. Overall, the workflow presented here comprises several improvements in the determination of protein lifetimes with respect to other available methods, including a minimally invasive labeling strategy and a robust interpretation of MS results, thus enhancing reproducibility across laboratories.

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Language(s): eng - English
 Dates: 2019-11-04
 Publication Status: Published online
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 Rev. Type: Peer
 Identifiers: DOI: 10.1038/s41596-019-0222-y
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Title: Nature Protocols
Source Genre: Journal
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Pages: - Volume / Issue: 14 (12) Sequence Number: - Start / End Page: 3333 - 3365 Identifier: -